This research program investigates immune responses induced by viral and plasmid vectors. Results are relevant to vaccine potency and mechanisms of actiond. In addition, immune responses can alter the safety and efficacy of gene therapy using viral or plasmid vectors, especially in the context of readministration, either by blocking therapy or by causing immunopathology. Better understanding of such immune responses can help select predictors of clinical success as well as adverse events, and thus contribute to improved regulatory decision-making. Our work focuses on the mouse influenza system. Main projects: a) Heterosubtypic immunity: Animals immunized against influenza A virus have cross-protection against challenge with flu of different subtypes. Vaccine development and prevention of pandemics would be aided by a more complete understanding the broad cross-protection against widely divergent viral strains that is observed in animals. We have extended our previous studies by more detailed analysis of depletion of T cell subsets during the challenge period. b) Immune responses induced by plasmid DNA: We are studying mechanisms responsible for immune protection by DNA vaccines encoding influenza virus antigens. Surprisingly, our results suggest that classical CTL are not required for protection. Flow cytometry and analysis of cytotoxic T cell activity are being used to further explore these findings. c) Many vaccines based on T cell immunity (HIV, hepatitis, flu) have been proposed when antibodies are ineffective or too narrow in viral strain specificity, but the protective efficacy of T cells acting in the absence of antibody has not been tested in most cases. Using mice lacking all antibodies due to disruption of Ig H and L chain genes, we have shown protection against influenza challenge, and shown that optimal protection requires both CD4+ and CD8+ T cells. We are now extending those studies into heterosubtypic systems. d) The role of IgA secretory antibodies in cross-protection is only incompletely understood. We are performing mucosal immunizations and challenges in knockout mice lacking IgA, to analyze the role of IgA in cross-protection, especially of the upper respiratory tract.